Our current research focus is driven by two general hypotheses. First, y-tubulin is responsible for anchoring microtubules in a specific, polarized orientation through precise interactions with microtubule subunits and with the Spc97p/Spc98p subunits of the spindle pole. Second, y-tubulin is involved in the mitotic checkpoint pathway apart from its role in nucleating microtubule assembly from the spindle poles. The first hypothesis is based on previous work by others and us. The second hypothesis is based on our collection of conditional alleles of human and S. pombe y-tubulin. Normally, abnormalities in spindle assembly arrest cells in prometaphase, and entry into anaphase and subsequent cytokinesis is inhibited. Unexpectedly, a subset of our y-tubulin mutants with abnormal spindles does not arrest but proceeds through anaphase and cytokinesis. Among the most striking are three cold sensitive mutant alleles. Two of the conditional mutants can fully rescue the lethal deletion of the endogenous S. pombe gamma-tubulin at the permissive temperature. Furthermore, we have isolated a mutant allele of rad2l+, a sister chromatid cohesin subunit encoding gene, as a suppressor of a gamma-tubulin mutant allele. Our progress in defining the molecular mechanisms of this novel gamma-tubulin function includes cloning of a heat sensitive mutant sgtl (suppressor of gamma TUbulin) and a related gene (46 percent similar protein product), sgt2+. Sgtlp and Sgt2p each have two protein-protein interaction mediating WW domains. Importantly, a multicopy suppressor of SGT1DELTA encodes a novel protein, Ssglp, and both y-tubulin and Ssglp contain putative WW domain-binding motifs. Our specific aims are, 1) to define the precise nature of y-tubulin interactions with microtubules and the spindle pole, 2) to determine if y-tubulin mutant alleles abrogate the mitotic checkpoint, 3) to investigate the molecular mechanism of the observed genetic interaction between gamma-tubulin and Sgtlp/Sgt2p, and 4) to define the mechanistic link between gamma-tubulin and the mitotic checkpoint pathways. We propose a combination of genetic, biochemical, and cellular approaches to achieve these aims. Each of the specific aims of this proposal is well founded in our recent progress and our progress in the past grant period. Our recent progress has put us in a position to make unique contributions to the mechanistic understanding of ytubulin functions during mitosis.